Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.
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We thank B. Kingston, C. Vakoc, M. Tolstorukov and G. Hannon for guidance and discussions, B. Bernstein, K. Plath, K. Chronis, Y. Shen and O. Tam for advice on the ATAC-seq analysis, P. Brown for providing the Dot1l inhibitor, B. Stillman for sharing the Chaf1b antibody and T. Graf for sharing the C10 cell line. We thank C. Nakada and Y. Kiyota (Nikon) for providing software to quantify iPS cell formation and A. Huebner for help with transdifferentiation experiments. We are grateful to H. Hock and the HSCI-CRM flow cytometry core for help with flow data analysis and to W. Mallard for initial RNA-sequencing analysis. We further thank B. Ma, S. Muller, M. Weissenboeck and the IMP/IMBA Biooptics and Transgenic core facility as well the CSF NGS laboratory for technical assistance and all members of the Hochedlinger, Zuber, Penninger, Elling, Shi and Kingston laboratories for their feedback on various aspects of this project. We thank A. Stark, A. Deaton and L. Barrett for critical reading of the manuscript. S.C. was supported by the PRCRP at the Department of Defense (CA 120212). H.Y.C. by was supported by NIH P50-HG007735. U.E. was funded by grants from IMBA and the Austrian National Foundation. S.W.L. was supported by a cancer center support grant and program project grant from the NCI and is an HHMI investigator. J.M.P. was supported by IMBA, ERC GA (number 341036) and the Innovator Award/Era of Hope Award Number W81XWH-12-1-0093. J.Z. was funded by an ERC starting grant (number 336860) and generous institutional funding from Boehringer Ingelheim. K.H. was supported by funds from the MGH, HHMI, NIH (R01 HD058013-06) and the Gerald and Darlene Jordan Chair in Regenerative Medicine.
The authors declare no competing financial interests.
Extended data figures and tables
a, Quantitative RT–PCR analysis to confirm suppression of Chaf1a and Chaf1b expression with miR-30-based vectors from arrayed screen. Sh Chaf1a pool, sh Chaf1b pool and sh CAF-1 pool denote pools of shRNAs targeting either Chaf1a, Chaf1b or both. b, Western blot analysis to confirm knockdown of CAF-1 components using the top-scoring miR-30-based shRNAs from arrayed screen (see Supplementary Fig. 1 for full scans). c, Quantification of data shown in Fig. 1f. d, Quantitative RT–PCR analysis confirming knockdown with top-scoring miR-E-based shRNAmiRs targeting Chaf1a, Chaf1b or Ube2i from the multiplexed screen. Error bars show s.d. from biological triplicates. RNA and protein were extracted from reprogrammable MEFs 72 h after doxycycline induction in panels a–d. e, Suppression of CAF-1 components, Ube2i and Setdb2 enhances reprogramming in the presence or absence of ascorbic acid (AA) as well as in serum replacement media containing LIF (SR-LIF). Oct4–GFP+ cells were scored by flow cytometry on day 11 after 7 days of OKSM induction and 4 days of transgene-independent growth. Error bars show s.d. from biological triplicates. f, Number of doxycycline-independent, alkaline phosphatase (AP)-positive colonies emerging two weeks after plating 10,000 reprogrammable MEFs carrying shRNA vectors against indicated targets and cultured in serum replacement media containing 2i (SR-2i), n = 1 experiment. g, Effect of suppressing SUMO E2 ligase Ube2i, E1 ligases Sae1 and Uba2 on iPS cell formation. Shown is fraction of Oct4–GFP+ cells at day 11 (7 days of OKSM induction, 4 days of transgene-independent growth). Error bars depict s.d. from biological triplicates.
Extended Data Figure 2 Germline transmission of iPS cells, genetic interaction of shRNA hits and effect of CAF-1 or Ube2i suppression on reprogramming dynamics.
a, Germline transmission of agouti chimaeras generated from iPS cells using doxycycline-inducible shRNA vectors targeting Chaf1a, Chaf1b or Ube2i. Germline transmission was determined by scoring for agouti coat colour offspring upon breeding chimaeras with albino females. Germline transmission was observed in 8/8, 4/4 and 6/8 cases for Chaf1a iPS-cell-derived chimaeras, in 7/7, 4/4, 7/7 and 9/9 cases for Chaf1b iPS-cell-derived chimaeras, and in 5/5, 7/7 and 5/5 cases for Ube2i iPS-cell-derived chimaeras. b, Table summarizing effects of co-suppressing pairs of targets on emergence of Oct4–GFP+ cells, shown as the ratio of Oct4–GFP+ to Oct4–GFP− cells relative to an empty vector control. Experiment equivalent to Fig. 2b except that second shRNAs were transduced two days after induction of reprogramming. c, Representative FACS plots showing effects of Chaf1a/b or Ube2i suppression on emergence of Oct4–GFP+ cells at days 7, 9 and 11 of OKSM expression. Histogram plots show fraction of Nanog+ cells within Oct4–GFP+ cells.
Extended Data Figure 3 Effect of CAF-1 suppression on OKSM levels and cellular growth, and shRNA rescue experiment.
a, Quantitative RT–PCR for transgenic OKSM expression using reprogrammable MEFs transduced with indicated shRNA vectors. Error bars show s.d. from biological triplicates. b, RNA-seq analysis of OKSM transgene expression in reprogrammable MEFs transduced with Renilla and Chaf1a shRNAs and exposed to doxycycline for 0, 3 or 6 days. Error bars indicate s.d. from biological triplicates. c, Western blot analysis for Sox2 and Tbp (loading control) in reprogrammable MEFs transduced with shRNA vectors targeting Renilla (Ren.713) or different CAF-1 components and exposed to doxycycline for 3 days (see Supplementary Fig. 1 for full scans). The same membrane was probed with anti-CAF-1 p150 and anti-CAF-1 p60 antibody to confirm knockdown (data not shown). d, Rescue experiment to demonstrate specificity of Chaf1b.367 shRNA vector. Reprogrammable MEFs carrying Oct4–tomato knock-in reporter were infected with lentiviral vectors expressing either EGFP or human CAF-1 p60 (CHAF1B) before transducing cells with Renilla or Cha1fb.367 shRNAs and applying doxycycline for 6 days. Colonies were counted at day 11. Note that CAF-1 p60 overexpression attenuates enhanced reprogramming elicited by Chaf1b suppression. e, f, Competitive proliferation assay between shRNA vector-infected and non-infected reprogrammable cells using indicated shRNAs in the presence or absence of doxycycline (OKSM expression). Note that CAF-1 suppression does not substantially affect the proliferation potential of reprogrammable MEFs after 1–3 days of doxycycline (OKSM) induction while it impairs the long-term growth potential of uninduced MEFs. Data were normalized to cell counts in ‘no OKSM’ condition for e and ‘day 2’ time point for f. Error bars show s.d. from biological triplicates.
Extended Data Figure 4 Confirmation of CAF-1 reprogramming phenotype with alternative transgenic and non-transgenic vector systems.
a, Alkaline phosphatase (AP)-positive, transgene-independent iPS cell colonies at day 14 following transduction of R26-M2rtTA MEFs with tetO-STEMCCA lentiviral OKSM expression vector and either Chaf1a.164 or Ren.713 shRNA vectors and treatment with high (2 μg ml−1) or low (0.2 μg ml−1) doses of doxycycline for 10 days. b, Quantification of data shown in a. Experiment was performed at 3 different plating densities (n = 1 experiment per density), representative data are shown. c, Comparison of reprogramming efficiencies between Col1a1::tetOP-OKSM; R26-M2rtTA reprogrammable MEFs and wild-type MEFs infected directly with OKSM-expressing lentiviral vectors containing either a strong Ef1a full-length promoter (Ef1a-OKSM long) or a weaker truncated promoter (Ef1a-OKSM short). TRE3G-OKSM is a lentiviral vector with a strong promoter, whose activity is downregulated over time upon infection of CAGS-rtTA3 transgenic MEFs (see below). Error bars show s.d. from biological triplicates. d, Quantitative RT–PCR data showing variability in OKSM expression levels over time using different vector systems. Cells were analysed after 3 and 6 days of infection (lentiviral vectors) or doxycycline exposure (reprogrammable MEFs). Error bars show s.d. from biological triplicates. OGR MEF, transgenic MEFs carrying Oct4–GFP and CAGS-rtTA3 alleles. e, Quantification of Oct4 protein levels by intracellular flow cytometry (top) and cellular granularity/complexity by side scatter (SSC) analysis of indicated samples (bottom). Error bars show s.d. from biological triplicates.
a, Competitive proliferation assay to determine effect of indicated Chaf1a and Chaf1b shRNA vectors on long-term growth potential of immortalized NIH3T3 cell line. Cells were infected with indicated constructs and the fraction of shRNA vector-positive cells was measured by flow cytometry at different time points. Data were normalized to cell counts at day 2 post-transduction. Rpa3.455, validated control shRNA targeting the broadly essential replication protein A3. Error bars show s.d. from biological triplicates. b, Histogram plots of MEFs harbouring R26-M2rtTA allele and either Col1a1::tetOP-miR30-tRFP-Ren.713 or Col1a1::tetOP-miR30-tRFP-Chaf1a.164 shRNA knock-in allele after transduction with pHAGE (Ef1a-OKSM) lentiviral vector and exposure of cells to different doses of doxycycline for 2, 4 and 6 days. Low doses of doxycycline (0.2 µg ml−1) result in lower expression of the shRNA miR cassettes than high doses of doxycycline (2 µg ml−1). c, Quantification of data shown in b using the geometric mean (n = 1 experiment for 3 indicated time points). d, Reprogramming efficiency of Col1a1::tetOP-miR30-tRFP-Chaf1a.164; R26-M2rtTA MEFs infected with pHAGE (Ef1a-OKSM) vector and induced with high (2 μg ml−1) or low (0.2 μg ml−1) doses of doxycycline for indicated number of days before scoring for Nanog+ iPS cells by immunocytochemistry on day 9. e, Classification of CRISPR/Cas9-induced mutations by sequence analysis of representative iPS cell clones (wt, wild type; indel, insertion/deletion; fs, frameshift; *, point mutation). f, Western blot analysis for CAF-1 subunits p150 and p60 in 6 representative iPS cell clones after CRISPR/Cas9-induced modifications of the Chaf1a locus (see Supplementary Fig. 1 for full scans). Wt/wt samples show unmodified wild-type control samples.
Extended Data Figure 6 Effect of CAF-1 suppression on HSP cell reprogramming and transdifferentiation.
a, Gating strategy for determining Pecam+ fraction (shaded area) in panel b; data identical to Fig. 4c. b, Quantification of the fraction of Pecam+ cells at day 4 and day 6 of reprogramming. Data obtained from one experiment using two different Chaf1 shRNAs. c, Transgene dependence assay during the reprogramming of haematopoietic stem and progenitor cells (HSP cells) into iPS cells in the presence of Chaf1a or Renilla shRNAs. Doxycycline pulses were given for 3 or 6 days and alkaline phosphatase (AP)-positive colonies were scored at day 10. d, Quantitative RT–PCR analysis of Chaf1a expression to confirm knockdown after 3 days of doxycycline induction, that is, coexpression of shRNAmiR and Ascl1 (n = 4 independent infections of the same Col1a1::tetOP-Chaf1a.164 shRNA MEF line; mean value ± s.d.). e, Gating strategy for determining Cd14+ and Mac1+ fractions (shaded area) shown in f; data identical to Fig. 4g. Positive gates were based on untreated (0 h) control cells. f, Quantification of the fraction of Cd14+ and Mac1+ cells at 0, 24 and 48 h of transdifferentiation using indicated CAF-1 shRNA or empty control vector (n = 2 independent infections; rep, replicate). g, Quantitative RT–PCR analysis of Chaf1a and Chaf1b expression to confirm knockdown in transduced pre-B cell line before induction of transdifferentiation (kd/ctrl, knockdown/empty vector control; n = 1 experiment, representative of 2 independent infections).
a, Experimental outline and assays (SONO-seq, ATAC-seq, Sox2 ChIP-seq, H3K9me3 ChIP-seq, microarrays and RNA-seq) to dissect effect of CAF-1 suppression on chromatin accessibility, transcription factor binding, heterochromatin patterns and gene expression. Assays were performed either in early reprogramming intermediates (day 3) or throughout the reprogramming time course (ATAC-seq and gene expression). b, SONO-seq analysis of CAF-1 knockdown and control cells at day 3 of reprogramming to determine accessible chromatin regions across promoters (n = 5,513) and ES-cell-specific enhancers (n = 14,265). CAF-1 shRNA vectors Chaf1a.164, Chaf1a.2120, Chaf1b.365 and Chaf1b.1221 were pooled for this experiment. c, ATAC-seq peak distribution across different genomic features. Shown is classification of peaks that are gained in CAF-1 knockdown cells compared to Renilla. d, ATAC-seq analysis of Chaf1a and Renilla control cells at day 3 of reprogramming to measure global chromatin accessibility over pluripotency-specific super-enhancer elements. ATAC-seq data from Chaf1a.164 shRNA- and Chaf1a.2120 shRNA-transduced cells were merged for this analysis. e, ATAC-seq accessibility maps at super-enhancer elements associated with the Sall4 locus. Shaded grey bars highlight more accessible sites in Chaf1a knockdown samples at days 3 and 6 of reprogramming compared to Renilla shRNA controls. f, ATAC-seq analysis of Chaf1a and control cells at day 3 of reprogramming to measure global chromatin accessibility over lineage-specific super-enhancer elements43 (C2C12, myoblast cell line; proB, progenitor B cells; Th, T helper cells). The n denotes number of examined enhancer elements for each cell type. ATAC-seq data from Chaf1a.164 shRNA- and Chaf1a.2120 shRNA-transduced cells were merged for this analysis.
a, Sox2 ChIP-seq enrichment across pluripotency-specific super-enhancer elements at day 3 of reprogramming in the presence of indicated shRNA vectors. b, Venn diagram depicting shared and unique Sox2 targets in Chaf1a and Renilla knockdown cells. c, Bar graph shows the number and fraction of ES-cell-specific Sox2 targets (blue colour) among Sox2-bound sites that are unique to Chaf1a or Renilla knockdown cells at day 3 of OKSM expression. d, Sox2 ChIP-seq analysis of Chaf1a and control shRNA-infected cells at day 3 of reprogramming to determine enrichment of Sox2 binding across lineage-specific super-enhancer elements43 (C2C12, myoblast cell line; proB, progenitor B cells; Th, T helper cells; P value <10−15 for all comparisons between Chaf1a knockdown cells and control).
Extended Data Figure 9 CAF-1 suppression induces specific depletion of H3K9me3 at somatic heterochromatin domains.
a, Scatter plots comparing H3K9me3 enrichment nearby ATAC-seq sensitive and super-enhancer regions between control (Ren.713) and Chaf1a knockdown cells (Chaf1a.164 and Chaf1a.2122) at day 3 of reprogramming. Values reflect normalized H3K9me3 ChIP signal (IP/input) for 5-kb genomic regions overlapping ATAC-seq sensitive regions (red), super-enhancer regions (orange) and regions within 50-kb upstream and downstream of super-enhancers (black). b, Scatter plots comparing H3K9me3 enrichment over transposable element (TE) families in control and Chaf1a knockdown cells at day 3 of reprogramming. Values reflect normalized H3K9me3 ChIP-seq signal (IP/input) over families of TEs in the mouse genome. c, Heatmap shows the relative changes (z-normalized) of TE family expression as estimated by RNA sequencing in control and Chaf1a knockdown cells at day 0, 3 and 6 of reprogramming. Data are clustered using the k-means algorithm. d, Cumulative histogram showing the relative fraction of reprogramming-resistant regions (RRRs)29 (x axis) that display negative or positive enrichment (fold change) of average H3K9me3 signal at day 3 of reprogramming in control and Chaf1a knockdown cells. Note that more RRRs exhibit depletion of H3K9me3 in Chaf1a knockdown samples. e, H3K9me3 ChIP-seq analysis of RRRs after 0 and 3 days of reprogramming. Box plots depict representative RRRs on chromosome 7 (P < 0.05 for both shRNAs). See also Fig. 5d. f, Histogram plot showing activation of UAS–Oct4–GFP transgene upon suppression of Chaf1b (shRNA+ line) in the presence of Gal4–VP16 fusion protein. See Fig. 5f for quantification.
This file contains full scans of Western Blot data for Figures 1f and Extended Data Figures 1b, 3c and 5f. (PDF 1421 kb)
This file contains Primary data from the arrayed shRNAmiR screen including all 1,075 tested shRNAmiRs, their sequences, corresponding scores for each biological replicate (FC replicate A/B) and their average (FC avg), which are plotted. (XLSX 130 kb)
This file contains primary data from the multiplexed shRNAmiR screen. Sheet 1 includes all 5,049 tested shRNAmiRs, their sequences and corresponding average scores for each time point. Sheet 2 shows raw data as normalized reads for all biological replicates (3-A01-D12, 48 replicates with OKSM induction after 3 days of G418 selection; 6-A01-D12, 48 replicates with OKSM induction after 3 days of G418 selection). Fold-change (FC) ratios for each shRNA were calculated by dividing the normalized reads of each shRNA by the normalized reads in MEFs 3 days after viral transduction. The mean of all FC values for the day 3 and day 6 time point were used to calculate the average FC for both time points (AvgFC_D3/6). In addition, FC ratios were used to calculate scores for each shRNA in each replicate (default score=0; score=1 if FC>1, score=3 if FC>10), which were summed up over all replicates separately for the day 3 and day 6 time point (Score_D3/6). (XLSX 2875 kb)
This file shows accessible super-enhancers in reprogramming intermediates upon CAF-1 suppression. Genomic coordinates of significantly more accessible super-enhancer (SE) sites in CAF-1 knockdown samples compared to Renilla control including log2 fold-enrichment scores (log2FC) and p-values for ATAC-seq data with indication of neighboring genes. (XLSX 12 kb)
This file shows correlation analysis between Sox2 binding sites and chromatin accessibility at super-enhancer sites in control and CAF-1 knockdown cells expressing OKSM. Sheet 1 shows Sox2 binding site counts in reprogramming intermediates (day 3) containing Chaf1a or Renilla shRNAs (unique and shared sites), ESC-specific sites, overlap with super-enhancers and positive correlation with ATAC-seq data. Sheet 2 depicts unique Sox2-bound super-enhancer sites in Chaf1a knockdown cells that are also more accessible by ATAC-Seq analysis, including genomic coordinates, neighboring genes and log2 fold-enrichment for both ChIP-seq and ATAC-seq data. (XLSX 10 kb)
This file shows significantly up-regulated genes in CAF-1 shRNA transduced MEFs relative to Renilla control by day 6 of reprogramming. Sheet1 contains genes that are either (1) significantly up-regulated at day 6 compared to day 3 in CAF-1 knockdown cells relative to Renilla control as determined by expression array data, or (2) significantly up-regulated at day 6 in CAF-1 shRNA transduced MEFs compared to Renilla control by RNA-seq analysis. (XLSX 9 kb)
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Cheloufi, S., Elling, U., Hopfgartner, B. et al. The histone chaperone CAF-1 safeguards somatic cell identity. Nature 528, 218–224 (2015). https://doi.org/10.1038/nature15749
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